The goal of the proposed research is to develop new methodologies for the genetic manipulation of B. pseudomallei. The tools to be developed should make it easier to create targeted mutations and multiple mutant strains, to identify unknown mutations not due to transposon insertion, to carry out complementation analysis for any region of the genome, and to generate and define strains with hybrid genomes. The proposed research is consistent with the exploratory/development nature of the R21 funding mechanism because it is designed to develop procedures with potentially broad impact on reaching the goals of the program announcement (PA-03-080), such as identifying B. pseudomallei virulence determinants and analyzing how they function, specifying innate and acquired antibiotic resistance mechanisms, identifying bacterial antigens recognized by the host, and creating live vaccine strains. [unreadable] [unreadable] [unreadable]